組織記憶依賴于遠端未受損區(qū)域的干細胞啟動Tissue memory relies on stem cell priming in distal undamaged areas

naturecellbiology

Article

/10.1038/s41556-023-01120-0

Tissuememoryreliesonstemcellprimingindistalundamagedareas

Received:16May2022

Accepted:28February2023

Publishedonline:20April2023

Checkforupdates

ChiaraLevraLevron

o

1,2,7,MikaWatanabe

c

1,2,7,ValentinaProserpio

o

1,2,3,7,GabrielePiacenti

c

1,2,AndreaLauria

o

1,2,3,StefanKaltenbach4,

AnnalauraTamburrini1,2,3,TakumaNohara5,FrancescaAnselmi1,2,

CarlottaDuval1,2,LucaElettrico1,2,DanielaDonna1,2,LauraConti2,6,DenisBaev3,KenNatsuga

5,TzachiHagai4,SalvatoreOliviero1,2,3&GiacomoDonati1,2

Epithelialcellsthatparticipatedinwoundrepairelicitamoreefcient

responsetofutureinjuries,whichisbelievedtobelocallyrestricted.Hereweshowthatcelladaptationresultingfromalocalizedtissuedamage

hasawidespatialimpactatascalenotpreviouslyappreciated.We

demonstratethataspecifcstemcellpopulation,distantfromtheoriginalinjury,originateslong-lastingwoundmemoryprogenitorsresidingin

theirownniche.Notably,thesedistalmemorycellshavenottakenpartinthefrsthealingbutbecomeintrinsicallypre-activatedthroughpriming.Thiscellstate,maintainedatthechromatinandtranscriptionallevel,

leadstoanenhancedwoundrepairthatispartiallyrecapitulatedthroughepigeneticperturbation.Importantlywoundmemoryhaslong-term

harmfulconsequences,exacerbatingtumourigenesis.Overall,weshowthatsub-organ-scaleadaptationtoinjuryreliesonspatiallyorganized

memory-dedicatedprogenitors,characterizedbyanactionablecellstatethatestablishesanepigeneticfeldcancerizationandpredisposesto

tumouronset.

Formingtheouterlayeroforgans,epitheliahavepredominantlyabar-rierfunctionandareabletosenseandadapttoenvironmentalchanges.Thehomeostaticintegrityofthesetissuesismaintainedthroughacontinuousturnoverensuredbystemcells(SCs)

1

–

3

,compartmental-izedasinthoseso-calledtransitionzones,presentintheepitheliaofoesophagus,eye,anus,lung,stomachandcervix

4

,

5

.Upontissuedam-age,eachepitheliallineageresidentnearbytheinjuryacquirescellplasticitythatallowscellstomigratetowardsthewoundsite,thuscontributingtothere-epithelialization

6

–

11

.

Inthepast6yearsitemergedthatepithelialcellsadapttoalocalstressfulevent,suchaswound,throughtheestablishmentofachro-matinmemorytorespondfastertoaneventualsimilarchallenge

12

,13

.

Nevertheless,thelineagespecificityofwoundmemories,throughadirectcomparisonofdifferentepidermalcellpopulations,hasnotyetbeenelucidated

14

.

ThepioneerworkbytheFuchslaboratoryshowedthattheSCslocatedincloseproximitytotheinjuredtissuecanbetrained,sug-gestingalocallyrestrictedpotentialofwoundmemory

12

,13

.However,besidesthepositiveeffectofmemoryonregenerativepotential

15

,negativeconsequencessuchascancer

16

mightberelatedtoit.Inthiscontext,itwouldbeoftranslationalinteresttounderstandthespatialdistributionandextentofwoundmemoryinepithelialcellslocateddistallyfromtherepairedareaandtocharacterizethefullimpactontheepithelium,longterm.

1DepartmentofLifeSciencesandSystemsBiology,UniversityofTurin,Torino,Italy.2MolecularBiotechnologyCenter‘GuidoTarone’,UniversityofTurin,Torino,Italy.3ItalianInstituteforGenomicMedicine,Candiolo(TO),Italy.4ShmunisSchoolofBiomedicineandCancerResearch,GeorgeSWiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel.5DepartmentofDermatology,FacultyofMedicineandGraduateSchoolofMedicine,Hokkaido

University,Sapporo,Japan.6DepartmentofMolecularBiotechnologyandHealthSciences,UniversityofTurin,Torino,Italy.7Theseauthorscontributedequally:ChiaraLevraLevron,MikaWatanabe,ValentinaProserpio.e-mail:

giacomo.donati@unito.it

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|Volume25|May2023|740–753741

Ithasbeendemonstratedthat,inparalleltotheimmunesystem,alsoepithelialcellsexhibittrainedwoundmemoryofaninjury.Afterawoundevent,thechromatinmemoryiskepttranscription-allydormant,butitallowsaquickre-activationintheeventofaneventualfurtherlesion

12

,

13

,

15

.Differently,anotheradaptationmecha-nismofimmunecells,namedpriming,describesanactivationstatethatneverturnsoffevenwhenthestimulusceases

17

.Currently,itisunknownifotheradaptationprogrammes,suchaspriming,areoptedbyepithelialcells

14

.

InthisArticle,inthecontextoftwoconsecutiveskininjuries,wecombinedlineagetracingwithsingle-cellanalysistocompre-hensivelyunderstandthespatialextentofwoundmemoryandthefullspectrumoftheadaptiveresponsesofepithelialcells(thatis,trainedwoundmemoryversuswoundpriming).Weshowthatspe-cificSCsgiverisetowound-primedprogenitorsthatexistinawideundamagedareadistantfromthedamagedzone,whileremainingwithintheiroriginalepidermalniche.Mechanistically,wedemon-stratethattranscriptionalderepressionisfunctionalformemoryonset.Finally,thememorythatisestablishedatthetranscriptionalandchromatinlevelinawideareasurroundingthewoundrepre-sentsanepigeneticfieldcancerizationevent

18

,

19

favouringtumouronset.Altogether,ourunexpectedresultsdrasticallychangetheassumptionrelativetothespatialdistributionofmemorycellsbyhighlightingtheexistenceofmemoryprogenitorslocatedfarfromtheinjury,inundamagedareas.

Results

TissueinjurieseducateSCsindistalundamagedareas

Recently,theexistenceofanepigeneticwoundmemoryhasbeenassessedforhairfollicle(HF)SCs

15

,butthepreciselineageidentity,thespatialdistributionandthespectrumofadaptationprogrammesacquiredbythememorycellsarestillunknown(Fig.

1a

).

Wefocusedonthreewell-characterizedcompartmentalizedcellpopulationsoftheHF:Lrig1+SCslocalizeattheHFjunctionalzone(JZ)andmaintainthesebocytes,sebaceousductsandinfundibulum(INFU);Gata6+arecommitted-to-differentiationanddifferentiatedductcellsoftheupperpilosebaceousunit;andLgr5+SCslocalizeatthelowerHF

11

,20

,21

.

Tounderstandiftheirprogenieselicitawound-inducedmemory,wegeneticallylabelled(GL)Lrig1+,Lgr5+andGata6+HFcells(ExtendedDataFig.1a,b)inadultmice.Weusedatwo-consecutive-injurymodelintailskin(Fig.

1b

)where,inthepresenceofaminimaltissuecon-tractionthesecondinjuryhealsfasterthanthefirstone(ExtendedDataFig.1c–f).Briefly,attime0wweperformedafirstfullthicknesswound.Eightweeksafter(8wpw1)whenanewhomeostasiswasreset(ExtendedDataFigs.1f–sand2a–e),weinducedasecondidenticalandoverlappinginjury(Fig.

1b

andExtendedDataFig.2f).ThisprocedureallowstheremovaloftheHF-derivedinterfollicularepidermal(IFE)SCsthatwerethefocusofGonzalesetal.’swork

15

.Inthesesettings,weinvestigatedthememoryofSCsthatremainlocalizedintheiroriginalHFniche,withoutcontributingtotherepairoftheIFE.

AlthoughthecontributionofLrig1GLcellsisquantitativelyhigherthanLgr5GLcells,bothprogeniesshowenhancedre-epithelializationabilityduringthesecondhealing,whiledifferentiatingGata6GLcellsdonot(Fig.

1c

andExtendedDataFig.2g).

Thespatialextentofthewoundmemoryisunknown,althoughthecellcontributiontoskinfullthicknesswoundrepairisspatiallyrestrictedtolessthan1mmawayfromtheinjuryina1mmearwoundcontext

22

,aswellasthecommunicationbetweendamagedHFs

23

.Consistently,thewound-engagedHFs(definedastheHFsinwhichGLtdTomato+cellsmovefromtheirhomeostaticHFnicheintoIFE)aremainlylocalizedintheclosesurroundingsoftheinjuryat1wpw1.However,at1wpw2thisphenotypeexistsupto7mmawayfromtheinjurysite,exclusivelyforLrig1progeny(Fig.

1d–g

andExtendedDataFig.2h–k).Horizontalwhole-mountconfirmedthatLrig1GLcellsinwounddistalareasremainlocalizedintheirniche(upperHF)at1wpw1until8wpw1,whiletheyexitintotheIFEasbasalandsuprabasalonlyat1wpw2(Fig.

1g

).Thiswound-elicitededucationofLrig1GLcellsindistalHFswasconfirmedinanadditionalsettingwhereasecondinjurywasperformeddistallyfromthepreviouslyhealedarea(zoneB)(Fig.

1d,h

andExtendedDataFig.2l,m).

Thus,weshowthat,asaconsequenceofalesion,differentepi-thelialSClineagesacquirememoryiflocatedinwoundproximity.However,exclusivelytheLrig1+SCprogenyiswoundeducatedwithintheHFslocatedupto~7mmfromtheinjury.Wewillrefertothisphe-nomenonasdistalmemory.

Distalmemoryelicitsanenhancedmigration

SinceLrig1GLcells,butnotLgr5GLones,showadaptivebehaviourtoinjuryindistalareas,wecomparedtheexpressionprofileoftheirsortedprogenies.Thetwohairfolliclestemcell(HFSC)lineageshavespecificwound-associatedtranscriptionalprogrammes(ExtendedDataFig.3a).Wedefinedthememorygenesasthosegenesderegu-latedduringthefirsthealingandwhosederegulationwasofgreatermagnitudeafterthesecondinjury.Lrig1GLcellshavemorememorygeneswithrespecttoLgr5GLcells(Fig.

2a

andSupplementaryTable1).TheirGeneOntology(GO)analysissuggests‘Cellpolarity/Migration’,amajorcellphenotypeinwoundhealing

22

,

24

,butnotproliferation,asafeatureofthewound-educatedLrig1GLcells(Fig.

2b

andExtendedDataFig.3b).

Tovalidatetheenhancedmigratorypotentialofdistalmemorycells,wecollectedskinbiopsiesat8wpw1fromdistalmemory(Distal)orna?ve,withoutmemory,(Ctrl)areasandweperformedmigrationassays

13

.ExvivomigrationassayconfirmedthehighermigratoryabilityofDistalLrig1GLcellswhencomparedwithCtrlaswellasanincreased

cellpolarization25

(Fig.

2c,d

andExtendedDataFig.3c–g).Thisisalsoconfirmedinvitro,inabsenceoftheirnichestimuli(Fig.

2e–g

andExtendedDataFig.3h–k).Thus,distalmemoryelicitsenhancedrepaircapabilitiesthat,onceestablished,aremaintainedintheabsenceofthephysiologicalmicroenvironment.

At8wpw1thememorygenesdisplay:(A)aconstitutiveexpres-sionpatternwheretheyremainderegulatedor(B)aninducibletrend

Fig.1|WoundmemoryinHFlineagesanditsspatialextension.

a,Left:repairingHFsinwoundproximityandtheHF-derivednewlyformed

epidermis,focusofthestudybyGonzalesetal.

15

.Right:cellsindistalHFsandtheiradaptationprogrammetowoundarethefocusofthisstudy.IFESCs:

interfollicularstemcells.b,Two-consecutive-skin-injurymodel:?1w,GL;0w,homeostasisandfirstinjury;1wpw1,1weekpostfirstwound;2wpw1,2weekspostfirstwound;8wpw1,8weekspostfirstwound,andsecondinjury;1wpw2,1weekpostsecondwound.c,LocalizationsofLrig1+,Lgr5+andGata6+cells

(left).Epidermalwhole-mountsofGLtdTomato+cells(redchannel)exiting

fromHFs(right).HG:hairgerm;INFU:infundibulum.d–g,Whole-mountsof

Lrig1GLengagedHFs(redchannel).Capitallettersdefinefourzones:A,upto1mmfromwound;B,1to3mm;Cfrom3to5mm;Dfrom5to7mm.DashedredlineshighlightrepresentativeengagedHFtriplets(d).NumberofengagedHFs.

n=6(0wand8wpw1),n=9(1wpw1and1wpw2)(e).Representativepictures

ofHFengagementinthefourzones(A,B,CandD)at1wpw1and1wpw2(f).

ImagesshowingthelocalizationofLrig1GLcellsinzoneD.AsterisksrepresentLrig1GLfibroblasts.WhitearrowindicatesGLcellsexitingintoIFE(g).h,Top:

epidermalwholemountsshowingtheclosureofasecondoverlappingwound

(1wpw2_Over)(left)orofadistalinjury(1wpw2_Distal),madeinzoneB(right).Bottom:quantificationofdistancefromwoundcentre.n=4(1wpw2_Over),n=5(1wpw1)andn=6(1wpw2_Distal).Yellowarrowsindicatewoundposition(fandg).Dashedcirclesindicatewoundperimeterat8wpw1(lightblue),0w(orange);linesunderlinethemigrationfrontofGLcellsat1wpw2(purple)or1wpw1

(green)(candh).P-valuefromatwo-tailedt-test.Dataaremean±s.d.Scalebars:

1mm(c,dandh);100μm(fandg).

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where,afterwoundresolution,the0wexpressionisrestored(Fig.

2h

andExtendedDataFig.3l,m).SinceinLrig1GLcellsalmost90%ofthememorygenesbelongtothe‘A’type,wehypothesizedthattheconstitutiveexpressionofmemorygenesat8wpw1mightbeduetotheexistenceofpriming,aswoundadaptationprogramme,inasubpopulationofLrig1SCprogeny.

WoundprimingofLrig1SCprogenyindistalHFs

Tobetterdissectthetranscriptionalbasisofwoundmemory,con-sideringcellheterogeneityinHFniches

26

,weperformedsingle-cellRNAsequencing(scRNA-seq)ofLrig1GLcellsat0w,1wpw1,8wpw1and1wpw2(Fig.

3a

andExtendedDataFig.4a).Thecombinationofunsupervisedclusteringanalysis,clustermarkeridentification,data

a

b

Knownandputativeadaptiveprogrammesafterinjury

Wound-distalHFs

Geneticlabelling

Woundsite

Firstinjury

–1w0w

Wound-trained

HF-derivedIFESCs

Trained

DistalHFSCsintheirnicheoforigin

memory

?

?

Trainedmemory

Priming

Nothing

Time

2wpw1

1wpw1

?

?

Tolerance

Second

injury

Firstinjury

Secondinjury

Second

injury

Firstinjury

Secondinjury

(Gonzalesetal.2021)

Firstinjury

(adaptedfrom

Divanghaietal.2021)

c

Lrig1GLcells

0w

8wpw1

1wpw2

1wpw1

1wpw2

8wpw1

d

Numberofengaged

ofHFs

1wpw1

8wpw1

1wpw1

pw2

1w

1

7

Wounddistance

(mm):

Wounddistance

(mm):1

7

Lgr5+

Discardofwound-trainedHF-derivedIFESCs

A

IFE

INFU

JZ

SG

e

B

GLcells

Lrig1

20

0w

1wpw1

8wpw1

1wpw2

0.0015

HF

HG/bulge

15

0.000002

C

Gata6+

0.002

10

IFE

0.000003

0.0002

D

0.005

5

INFB

0.008

JZ

SG

0

Wounddistance(mm):

HF

A

HG/bulge

1

7

Lrig1+

B

IFE

INFBSG

C

h

JZ

HF

D

HG/bulge

Lgr5GLcells

+

Gata6GLcells

Lrig1GLcells

1wpw2_over

1wpw2_distal

f

g

Lrig1GLcellsinD

C

A

B

D

Lrig1GLcellsKI67

1wpw1

1wpw2_over

1wpw2_distal

Distancefrom

woundcentre(mm)

1wpw1

1wpw2

0w

0.8

0.6

0.4

0.2

0

0.015

0.030

1wpw1

*

*

**

*

8wpw11wpw2

*

*

*

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a

Memorygenesdefinition

A

1wpw2

Down

Up

1wpw11wpw2

1wpw1

Lrig1GLcellsLgr5GLcells

1,08629430

MemorygenesoverDEG

WoundmemorygenesNon-memorygenes

c

b

Tissue/cellisolationforexvivoandinvitroassaysat8wpw1

GOcellularcomponent_Lrig1

DistalCtrl

Golgistack(GO:0005795)

Filopodium(GO:0030175)

A

A

Woundbed

Ruflemembrane(GO:0032587)

–log10(P)012

Distancefromwound:2–7mm>2cm

21%

47%

Day9Day7Day4

d

g

Exitlength(mm)

Ctrl

Distal

Ctrl

Distal

Ctrl

Distal

CtrlDistal

Lrig1GLcells_8wpw1

0.0135

3.0

0.0045

2.5

2.0

0.000008

1.5

1.0

0

Day4Day7Day9

GOanalysisrelativetoupregulatedgenesinvitro8wpw1_distal

FocaladhesionActincytoskeletonGlycolysis

–log10(AdjP)

0369

Fig.2|TranscriptomeofLrig1GLcellspredictsanewprimedsubpopulation

arisingafterwoundresolution.a,Memorygenes(logFC(1wpw2)>logFC

(1wpw1))andVenndiagramreportingthenumberandthepercentageof

inferredmemorygenesoverthetotalDEGsacrossthetimecourseinLgr5andLrig1GLcells.n=3mice.b,EnrichedGOtermsformemorygenesinLrig1GLcells,as?log10ofp-value.Dashedlineindicatessignificance.n=3mice.c,Exvivoandinvitroexperimentalsettingsat8wpw1:Lrig1GLskinbiopsiesfromwound-educatedDistalregionandfromCtrlarea,outsidefrommemoryzonearecompared.Forinvitroassaysthebiopsiesweredissociated,andcellswereplated.d,ImagesofexvivomigrationofLrig1GLcells(left)andquantificationofthemigration(exitlength)(right).Dashedlinesmarkthemigrationfront

Invitro

trackdisplacement(μm)

e

15

10

5

0

Ctrl

Distal

8wpw1

Lrig1GLcells

0.045

f

8wpw1

Lrig1GLcells

150

100

50

(μm)

–150–100100150

–50

Ctrl

(μm)–100Distal

h

Subclassificationofmemorygenes

8wpw1/0w

8wpw1/0w

1wpw2/0w

1wpw2/0w

ConstitutiveInducible

DEG

Lgr5GL

Lrig1GL

1wpw1/0w1wpw1/0w

ofepidermalcells.Scalebar:2mm.Dataaremean±s.e.m.n=9skinexplants.e,f,Timelapsemigrationassayinvitro.Trackdisplacement(μm)(e)and

representativenormalizedstartpositiongraph(f)ofculturedLrig1GLcells.

Dataaremean±s.d.n=4mice.g,RNA-seqofculturedLrig1GLcellsfrom

wound-educateddistalregion(Distal3–7mmfromwoundbed(n=6))andCtrl(distance>2cmfromwoundbed(n=8)).TheGOanalysisrelativetoupregulatedgenesinDistalisreported,as?log10ofadjustedp-value(AdjP).Dashedline

underlinessignificance.h,Circularideogramplot(CIRCOS)ofsharedDEGs

(grey)between1wpw1,8wpw1and1wpw2(green)orbetween1wpw1and1wpw2only(yellow),inLgr5andLrig1GLskins.P-valuefromatwo-tailedt-test.

integrationwithJoostetal.

26

dataset,togetherwithpseudotimeanalysisdistinguishescellsaccordingtolineageidentityanddifferentiationstage(Fig.

3b–d

,ExtendedDataFig.4b–fandSupplementaryTable2)issummarizedinFig.

3c

.Aspreviouslysuggested

20

,ourscRNA-seqdataintegratedwithmarkergenesfromDekonincketal.

27

confirmthatLrig1GLcellscontributetothehealingspecificallyasinterscalelineage,oneofthetwoIFEdifferentiationprogrammes

28

(ExtendedDataFig.4g).

TrajectoryDisthemostinterestingintermsofwound-inducedplasticity.Indeed,Lrig1+SCsacquireplasticitywhileleavingtheNicheofOrigin-JZCluster11andmovealongthetrajectoryintheTransitionCluster7towardsdifferentiatedIFE(cluster0),wheretheycontributetotherepair(Fig.

3e

).IntheTransitionCluster,cellsresideintheINFUat

thestartingpointofthetrajectoryandthenmovetowardsIFE,assug-gestedbytheexpressionoftheINFUmarkerPostn

26

(ExtendedDataFig.4h).Comparingthetwohomeostaticstages(0wand8wpw1),wenoticeanunexpectedincreaseinthenumberofLrig1GLcellsintheINFUat8wpw1(Fig.

3e,f

).Sincethecellsfromtheinfundibularclusterat8wpw1expressintermediatelevelsoftheTransitionClustergenes,between0wandhealingphases(1wpw1and1wpw2),withthesecondinductionbeinggreaterthan1wpw1(Fig.

3g

),weinfertheexistenceofprimingadaptiveprogramme,asdescribedforimmunecells

17

.Indeed,inthenewlyestablishedhomeostasis(8wpw1)thegenesassociatedwith‘Cellactivation’andthewound-activationmarker

29

Krt6areprimed(ExtendedDataFig.4h–j).

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a

0w

1wpw18wpw11wpw2

d

0

7

9

b

0

12

1

2

4

3

4

5

6

7

1

116

13

8

8

2

3

9

10

11

5

12

13

10

SG

Bulb

sebaceous

cIFE

interfollicularepidermis

INFU

infundibulum

Junctionalzone

JZstemcells

gland

JZandSGducts

Bulge

Diferentiation

PlotofgenegroupsfromJoostetal.CellSystems2016onLrig1scRNA-seq

SGOBIFE.DIGli1_groupKrt14_groupLrig1_groupLgr5_group

uHF.IuHF.IIuHF.III

15Norm-log(counts)

Averagez-score

e

g

Top100markersoftransitioncluster

12

Transitioncluster

4

0

TrajectoryD

0.0135

0.000004

7

3

2

Nicheoforigin

JZcluster11

1

Start

0

0w

1wpw1

–1

8wpw11wpw2

0w1wpw18wpw11wpw2

End

8wpw1_distal

Lrig1GLcells

*

0w

f

*

h

iSpatialexpressionoftop100markersoftransitioncluster

INFU_8wpw1

0.0000000045

0w8wpw1_distal

KRT6

Lrig1GLcells

0.0022

8wpw1_Ctrl

0.06

KRT6intensity(AU)

Averagez-score

0.0002

8wpw1_distal2.0

8wpw1_Wb

10

(ratioto0w)

1.5

1.0

0.0045

5

0.5

0

0

UndiferentiatedSca1+GLcells

WbDistalCtrl

–0.5

WbDistalCtrl

Fig.3|Identificationofwound-primedcellsintheINFUwithapre-activated

transcriptionalprogramme.a,UMAPofscRNA-seqdataofLrig1GLcellsat0w(red),1wpw1(green),8wpw(lightblue)and1wpw2(purple).b,Unsupervised

clusteringofsingle-celltranscriptomicdata.c,SummaryillustratingepidermallineagesanddifferentiationinLrig1GLsingle-celldata.Cellsarecoloured

accordingtotheirepidermallineages.Dashedlineidentifiesthehomeostatic

compartmentboundarybetweenupperandlowerHF

11

.d,Expressionplotof

genesetfromJoostetal.study

26

.e,Pseudotimeanalysis.TrajectoryDiscolouredbytimepointsandclusters.f,EpidermalwholemountsofLrig1GLtdTomato+

cellsoccupancyshowingcelllocalizationintheINFU(whitearrows)inthe

distalHFsat8wpw1(at5mmfromwoundsite).AsterisksmarkdifferentiatedIFEcells.g,Plotoftheaveragez-scoreofthetop100markersofTransition

Cluster7showingtheintermediatetranscriptionalstateat8wpw1.h,INFU

whole-mountpicturesofKrt6at0wand8wpw1at5mmfromwoundsite(left)

andquantificationat8wpw1(right),inwoundbed(Wb),distalmemoryregion

(Distal3–7mmfromWb)orfromana?veregion(Ctrl>2cmfromWb).Ratioto

0wisplotted.Dashedlinesindicate0waverage.Dataaremean±s.d.n=3mice.i,SpatiallyresolvedscRNA-seqanalysisofupper-HFLrig1GLcellsfromwound

bed(Wb),distalmemoryregion(Distal3–7mmfromWb)andna?veregion(Ctrl>2cmfromWb)wasperformedat8wpw1andanalysedinExtendedDataFig.5.

Plotofaveragez-scoreexpressionofthe100markersoftheTransitionCluster

identifiedinFig.

3g

inthecellsfromeachgroup.n=42(Wb),n=48(Distal),n=59(Ctrl)cells.P-valuefromatwo-tailedt-test.Scalebars:100μm(fandh).Dataaremedianwith25thand75thpercentilesifnotdifferentlyindicated.scRNA-seq

data(a–e,gandi)aretheintegrationoftwoindependentexperiments,eachofthembasedonfourbiologicalreplicates.

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a

b

TrajectoryD

0w

1wpw18wpw11wpw2

0

12

7

Transitioncluster

11

NicheoforiginJZcluster

Cellplasticity

Timepoint

genes

Cluster

1.0

Cluster0.50011–0.57

12

EMT

Timepoint0w

1wpw18wpw11wpw2

ExtracellularmatrixorganizationFocaladhesionHypoxia

Glycolysis

mTORC1signallingActincytoskeletonMitoticspindle

SRE

–log10(AdjP)

0510

–11

d

cINFU_0wINFU_8wpw1_distal

BM

0.0460.00190.00170.0270.001

0.5

logratio(height/width)

0

–0.5

–1.0

–1.5

Lrig1GLtdTomato+basalcellsinINFU

versus

0.046

1wpw1

1wpw2

8wpw1

0wWbDistal1Distal2

Lrig1GLcellsPhalloidinBasementmembranereference

e

4

Glut1z-score

2

0

–2

f

TransitionclusterAllbut

Cluster11Cluster7Cluster0Cluster12clusters

20

11,7,0,12

GLUT1intensity(AU)

15

10

5

0

0w1wpw18wpw11wpw2

INFU_8wpw10.021

WbDistalCtrl

g

0w8wpw1_distal

Trackdisplacement(μm)

60

40

30

20

10

0

GLUT1

Lrig1GLcells

Lrig1GL

cells_8wpw1

0.009

GLUT1–GLUT+

Fig.4|Spatialdistributionandcellstatecharacterizationofpriming.

a,UMAPofpseudotimetrajectoryDwithtimepoint(top)andclusters(bottom).b,Comparisonoffirstandsecondhealing.Left:smoothedrelativeexpression

(SRE)ofderegulatedgenesintrajectoryD.Clustersandtimepointsareindicatedabove,andtransientlyinducedcellplasticitygenes(SupplementaryTable3)

arehighlighted(blackbox).Right:averagez-scoreexpressionofcellplasticitygenes(top)andtheirGOanalysis(bottom)as?log10oftheadjustedp-value

(AdjP).Dashedlineunderlinessignificance.c,Singlestackofepidermalwhole-mountstainedwithphalloi